Unacylated ghrelin as therapeutic agent in the treatment of metabolic disorders

ABSTRACT

An isolated polypeptide comprising any amino acid fragment of unacylated ghrelin or any analog thereof, wherein the polypeptide has an activity selected from the group consisting of a) decreasing blood glucose levels; b) increasing insulin secretion an/or sensitivity; c) binding to insulin-secreting cells; and d) promoting survival of insulin-secreting cells. As well as the use of the polypeptide in the treatment of a disorder associated with impaired glucose metabolism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisionalpatent application No. 60/941,186 filed May 31, 2007, the content ofwhich is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to unacylated ghrelin fragments and analogsthereof as well as to their therapeutic uses.

BACKGROUND

Ghrelin is a peptide which was isolated from the stomach but isexpressed also in many other tissues, including the endocrine pancreas.It was discovered as a natural ligand of the growth-hormone secretagoguereceptor type 1a (GHS-R) (Refs. 1, 2). Ghrelin acylation at serine 3 isessential for binding to GHS-R1a, which mediates GH-releasing activityand also the orexigenic action of acylated ghrelin. Besides stimulatingGH secretion and modulating other pituitary functions, acylated ghrelin(AG) exerts a broad range of biological actions such as centralregulation of food intake and energy balance and control of insulinsecretion and glucose metabolism. GHS-R1a expression has been detectedin a variety of endocrine and non-endocrine, central and peripheralanimal and human tissues, including the pancreas. Notably, the linkbetween ghrelin and insulin seems of major relevance. AG has been shownto possess hyperglycemic diabetogenic effects; ghrelin knock-out micedisplay enhanced glucose-induced insulin release while blockade ofpancreatic islet-derived ghrelin has been shown to enhance insulinsecretion and to prevent high-fat diet-induced glucose intolerance inrats.

In the endocrine pancreas, ghrelin has been shown to localize to α- andβ-cells and to the newly identified ghrelin-producing islet ε-cells,suggesting a role in the regulation of β-cell fate and function (Refs.9, 22, 19). Survival of β-cells is of major importance for maintainingnormal glucose metabolism and ft-cell apoptosis is a critical event inboth type 1 and 2 diabetes (Refs. 16, 21).

Unacylated ghrelin (UAG) is the major circulating form of ghrelin andhas long been believed to be biologically inactive since it does notbind GHS-R1a at physiological concentrations and is thus devoid ofGH-releasing activity. It is now known that UAG is a biologically activepeptide, particularly at the metabolic level, having notably been shownto exert anti-diabetogenic effects as described in U.S. patentapplication Ser. No. 10/499,376, published on Apr. 14, 2005, underpublication number US 2005-0080007. Indeed UAG is able to: a) counteractthe hyperglycemic effect of AG in humans (Ref. 6); b) directly modulateglucose metabolism at the hepatic level by blocking basal,glucagon-induced and acylated ghrelin-stimulated glucose output fromhepatocytes (Ref. 3); c) decrease fat deposition, food consumption, andglucose levels in UAG transgenic animals (Ref. 7); d) stimulateproliferation and prevent cell death and apoptosis in β-cells and humanpancreatic islets (Ref. 4).

It has recently been demonstrated that UAG is able to stimulateproliferation and to prevent cell death and apoptosis induced by(IFN)-γ/tumor necrosis (TNF)-α, synergism in β-cells and humanpancreatic islets (Ref. 4). Noteworthy, cytokine synergism is consideredto be a major cause for β-cell destruction in type I diabetes as well asof β-cell loss in type 2 diabetes. Moreover, this work also showed thatUAG stimulated glucose-induced insulin secretion from β-cells that donot express GHS-R1a.

Together, these results reinforce the concept that UAG has a therapeuticpotential in medical conditions associated with metabolic disorder suchas conditions characterized by insulin deficiencies or by insulinresistance, including, but not limited to diabetes, and the effect ofUAG on the β-cells is one of the mechanisms of action of UAG in thesepotential applications.

Recently, the therapeutic potential of UAG was clinically demonstrated,as a continuous infusion of UAG in healthy volunteers resulted in alowering of blood glucose, an improvement in insulin sensitivity, areduction in blood free fatty acids, and decreased cortisol levels.

UAG is a 28 amino-acid peptide and would preferably be administered topatients by intravenous or subcutaneous injection in order to produceits effects, which is not a convenient way to administer a drug to apatient. Also, peptides of this size are usually rapidly degradedfollowing administration and their in vivo efficacy is often weakfollowing intravenous, subcutaneous or intramuscular bolusadministration.

In addition, manufacturing a 28 amino-acid peptide is a long andexpensive process, whether it is manufactured by solid-phase peptidesynthesis or by recombinant technology. Finally, chronically treatingpatients with a long peptide such as UAG might represent safety risksfor the patients in the form of immunogenicity. Raising neutralizingantibodies against a natural peptide is a potential major health riskfor the patients.

Therefore, it would be highly desirable to identify smaller sizepeptides that would possess a comparable biological activity to UAG, butwould be easier and less costly to manufacture.

It would be even more desirable that these smaller size peptides wouldhave increased biological potency when compared with UAG.

Another advantage of these smaller size peptides would be that theywould bear fewer immunogenicity risks for patients upon chronic andrepeated administrations, and hence exhibit a better safety profile.They may have a better bioavailability than UAG, whatever the route ofadministration, and be suitable for more convenient routes ofadministration, such as, but not limited to, transdermal, pulmonary,intranasal or oral delivery, or may constitute a starting material forthe design of peptide analogs or peptidomimetic molecules with a betteroral bioavailability. Smaller size peptides may also be compatible withdrug delivery system such as, but not limited to, polymer-based depotformulations.

SUMMARY

In one aspect of the present invention, there is provided an isolatedpolypeptide comprising any amino acid fragment of the amino acidsequence shown in SEQ ID NO: 9 or an analog thereof, wherein saidpolypeptide has at least one activity selected from the group consistingof a) decreasing blood glucose levels; b) increasing insulin secretionand/or sensitivity; c) binding to insulin-secreting cells; and d)promoting survival of insulin-secreting cells.

In one aspect of the present invention, there is provided an isolatedpolypeptide 5 to 27 amino acid residues in length, said polypeptidecomprising the amino acid sequence Glu-His-Gln-Arg-Val.

In another aspect of the present invention, there is provided a methodfor treating a disorder associated with impaired glucose metabolism in apatient, comprising administering to the patient a therapeuticallyeffective amount of the polypeptide as defined herein.

In another aspect of the present invention, there is provided a methodfor enhancing survival and/or proliferation of insulin-secreting cellscomprising culturing said cells in the presence of a therapeuticallyeffective amount of the polypeptide as defined herein.

In another aspect of the present invention, there is provided use of atherapeutically effective amount of the polypeptide as defined herein,in the preparation of a medicament for treating a disorder associatedwith impaired glucose metabolism in a patient.

In a further aspect of the present invention, there is provided use of atherapeutically effective amount of the polypeptide as defined herein,for treating a disorder associated with impaired glucose metabolism in apatient.

In a further aspect of the present invention, there is provided apharmaceutical composition for treating a metabolic disorder associatedwith impaired glucose metabolism comprising a therapeutically effectiveamount of the polypeptide as defined herein.

In yet a further aspect of the present invention, there is provided anisolated polypeptide selected from the group consisting of SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO:28.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates survival of INS-1E β-cells in serum-free medium inthe presence of unacylated ghrelin or the indicated fragments ofunacylated ghrelin.

FIG. 2 illustrates survival of INS-1E β-cells in the presence ofTNF-α/IFN-γ/IL-β and in the presence of unacylated ghrelin or theindicated fragments of unacylated ghrelin.

FIGS. 3A and 3B illustrate survival of HIT-T15 β-cells in serum freemedium with or without cytokines and either unacylated ghrelin UAG(1-28) or its fragment UAG (1-14) (FIG. 3A) or UAG (1-18) (FIG. 3B).

FIGS. 4A and 4B illustrate survival of HIT-T15 β-cells in serum freemedium with or without cytokines and either unacylated ghrelin UAG(1-28) or its fragments UAG (1-5) (FIG. 4A) or UAG (17-28) (FIG. 4B).

FIGS. 5A and 5B illustrate survival of cytokine-treated HIT-T15 β-cellin the presence of unacylated ghrelin fragments UAG (6-13), UAG (8-13),UAG (8-12), UAG (1-14), UAG (1-18), UAG (1-28) (FIG. 5A) and UAG (8-11),UAG (9-12) and UAG (9-11) (FIG. 5B).

FIGS. 6A to 6C illustrate the antiapoptotic effects of unacylatedghrelin fragments UAG (6-13) (FIG. 6A), UAG (8-13) (FIG. 6B) and UAG(8-12) (FIG. 6C) on cytokine treated HIT-T15 β-cells

FIGS. 7A and 7B illustrate the survival effect on human pancreaticislets of unacylated ghrelin (1-28) and its fragments UAG (1-14), UAG(1-18) (FIG. 7A) and UAG (1-5) and UAG (17-28) (FIG. 7B).

FIGS. 8A to 8D illustrate the effect of UAG (1-14) (FIG. 8A), UAG (1-18)(FIG. 8B), UAG (1-28) (FIG. 8C) and Exendin-4 (FIG. 8D) on insulinsecretion in human pancreatic islets.

FIGS. 9A to 9D illustrate the in vivo effect of unacylated ghrelinfragment UAG (6-13) on animal survival (FIG. 9A), on plasma glucoselevels (FIG. 9B) and plasma (FIG. 9C) and pancreatic (FIG. 9D) insulinlevels, in Streptozotocin (STZ)-treated animals.

FIGS. 10A and 10B illustrate the binding of unacylated ghrelin andunacylated ghrelin fragment UAG (6-13) to pancreatic HIT-T15 (FIG. 10A)and INS-1E (FIG. 10B) β-cell receptors.

FIGS. 11A and 11B illustrate the survival effects of UAG (6-13) withalanine (Ala) substitutions at positions 6 to 13 in HIT-T15 β-cells inboth the absence of serum (FIG. 11A) and in the presence of cytokines(FIG. 11B).

FIGS. 12A and 12B illustrate the survival effects of UAG (6-13) withconservative substitutions and N-terminal modifications in HIT-T15β-cells, in both the absence of serum (FIG. 12A) and in the presence ofcytokines (FIG. 12B).

FIGS. 13A and 13B illustrate the survival effects of UAG (6-13) withcyclization in HIT-T15 β-cells in both the absence of serum (FIG. 13A)and in the presence of cytokines (FIG. 13B).

FIGS. 14A and 14B illustrate the in vivo effects of UAG (6-13) on plasmaglucose levels after 2 and 4 weeks of treatment in ob/ob mice, an animalmodel of diabetes associated with obesity. FIG. 14A illustrates fedplasma glucose levels and FIG. 14B illustrates fasting plasma glucose.

FIG. 15 illustrates fasting insulin levels after 2 and 4 weeks oftreatment with UAG and UAG (6-13) in ob/ob mice, an animal model ofdiabetes associated with obesity.

FIG. 16 illustrates the effect of UAG and UAG (6-13) on gonadal fat aspercent body weight in ob/ob mice, an animal model of diabetesassociated with obesity.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention belongs.

UAG Fragments and Analogs Thereof

For the purpose of the present invention the following terms are definedbelow.

In the present application, the terms “ghrelin” and “acylated ghrelin”or “AG” are used interchangeably and have the same meaning.

The term “unacylated ghrelin” or “UAG” is intended to mean peptides thatcontain the amino acid sequence specified in SEQ ID NO: 1(1-NH₂Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-28;SEQ ID NO: 1). UAG may also be referred to as UAG (1-28).

Naturally-occurring variations of unacylated ghrelin include peptidesthat contain substitutions, additions or deletions of one or more aminoacids which result due to discrete changes in the nucleotide sequence ofthe encoding ghrelin gene or alleles thereof or due to alternativesplicing of the transcribed RNA. It is understood that the said changesdo not substantially affect the properties, pharmacological andbiological characteristics of unacylated ghrelin variants. Thosepeptides may be in the form of salts. Particularly the acidic functionsof the molecule may be replaced by a salt derivative thereof such as,but not limited to, a trifluoroacetate salt.

As used herein, SEQ ID NO: 9 refers to the amino acid sequenceconsisting of residues 6 to 18 of UAG (SEQ ID NO: 1), namely:6-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-18.

By “peptide”, “polypeptide” or “protein” is meant any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation), or chemical modification, or thosecontaining unnatural or unusual amino acids such as D-Tyr, ornithine,amino-adipic acid. The terms are used interchangeably in the presentapplication.

The term “fragments” or “fragments thereof” refers to amino acidfragments of a peptide such as unacylated ghrelin. Fragments ofunacylated ghrelin are shorter than 28 amino acid residues. Fragments ofunacylated ghrelin may therefore be 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acidresidues in length.

In some aspects of the invention, the polypeptides are used in a formthat is “purified”, “isolated” or “substantially pure”. The polypeptidesare “purified”, “isolated” or “substantially pure” when they areseparated from the components that naturally accompany them. Typically,a compound is substantially pure when it is at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by weight, of the totalmaterial in a sample.

The term “analog of unacylated ghrelin”, “analog of fragments ofunacylated ghrelin” or “analogs thereof” refers to both structural andfunctional analogs of unacylated ghrelin or fragments thereof which are,inter alia, capable of replacing unacylated ghrelin in antagonizing theperipheral actions or functions of ghrelin or are capable of replacingother biological actions of unacylated ghrelin, such as, but not limitedto, stimulate proliferation and/or inhibit apoptosis in β-cell lines,lower blood glucose levels, improved insulin sensitivity and/orsecretion, decrease cortisol levels, improve lipid profile in humanbeings, and thus, have the potential use to treat metabolic disorderssuch as those associated with for example, insulin resistance, insulindeficiency, dyslipidemia or cortisol excess.

Simple structural analogs comprise peptides showing homology withunacylated ghrelin as set forth in SEQ ID NO: 1 or homology with anyfragments thereof. For example, an isoform of ghrelin-28 (SEQ ID NO: 1),des Gln-14 Ghrelin (a 27 amino acid peptide possessing serine 3modification by n-octanoic acid) is shown to be present in stomach. Itis functionally identical to ghrelin in that it binds to GHSR-1a withsimilar binding affinity, elicits Ca²⁺ fluxes in cloned cells andinduces GH secretion with similar potency as Ghrelin-28. It is expectedthat UAG also has a des Gln-14 UAG that is functionally identical toUAG.

Preferred analogs of UAG and preferred analogs of fragments of UAG arethose that vary from the native UAG sequence or from the native UAGfragment sequence by conservative amino acid substitutions; i.e., thosethat substitute a residue with another of like characteristics. Typicalsubstitutions include those among Ala, Val, Leu and Ile; among Ser andThr; among the acidic residues Asp and Glu; among Asn and Gln; among thebasic residues Lys and Arg; and among the aromatic residues Phe and Tyr.Particularly preferred are analogs in which several, for example, butnot limited to, 5-10, 1-5, or 1-2 amino acids are substituted, deleted,or added in any combination. For example, the analogs of UAG may differin sequence from UAG by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions (preferably conservative substitutions), deletions, oradditions, or combinations thereof.

There are provided herein, analogs of the peptides of the invention thathave at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence homology with the amino acid sequencesdescribed herein over its full length, and sharing at least one of themetabolic effects or biological activity of UAG. A person skilled in theart would readily identify an analog sequence of unacylated ghrelin oran analog sequence of a fragment of unacylated ghrelin.

In a further aspect, analogs of UAG or fragments thereof are, forexample, analogs obtained by alanine scans, by substitution with D-aminoacids or with synthetic amino acids or by cyclization of the peptide.Analogs of UAG or fragments thereof may comprise a non-naturally encodedamino acid, wherein the non-naturally encoding amino acid refers to anamino acid that is not one of the common amino acids or pyrrolysine orselenocysteine, or an amino acid that occur by modification (e.g.post-translational modification) of naturally encoded amino acid(including, but not limited to, the 20 common amino acids or pyrrolysineand selenocysteine) but are not themselves incorporated into a growingpolypeptide chain by the translation complex. Examples of suchnon-naturally-occurring amino acids include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine andO-phosphotyrosine.

As used herein, the term “modified” refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide.

The term “post-translational modification” refers to any modification ofa natural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as incell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications. Examples ofpost-translational modifications are, but are not limited to,glycosylation, acetylation, acylation, amidation, carboxylation,phosphorylation, addition of salts, amides or esters, in particularC-terminal esters, and N-acyl derivatives of the peptides of theinvention. The types of post-translational modifications are well known.

Certain peptides according to the present invention may also be incyclized form, such that the N- or C-termini are linked head-to-taileither directly, or through the insertion of a linker moiety, suchmoiety itself generally comprises one or more amino acid residues asrequired to join the backbone in such a manner as to avoid altering thethree-dimensional structure of the peptide with respect to thenon-cyclized form. Such peptide derivatives may have improved stabilityand bioavailability relative to the non-cyclized peptides. Methods forcyclizing peptides are well known in the art.

Cyclisation may be accomplished by disulfide bond formation between twoside chain functional groups, amide or ester bond formation between oneside chain functional group and the backbone α-amino or carboxylfunction, amide or ester bond formation between two side chainfunctional groups, or amide bond formation between the backbone α-aminoand carboxyl functions. These cyclisation reactions have beentraditionally carried out at high dilution in solution. Cyclisation iscommonly accomplished while the peptide is attached to the resin. One ofthe most common ways of synthesising cyclic peptides on a solid supportis by attaching the side chain of an amino acid to the resin. Usingappropriate protection strategies, the C- and N-termini can beselectively deprotected and cyclised on the resin after chain assembly.This strategy is widely used, and is compatible with eithertert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc)protocols. However, it is restricted to peptides that containappropriate side chain functionality to attach to the solid support. Anumber of approaches may be used to achieve efficient synthesis ofcyclic peptides. One procedure for synthesising cyclic peptides is basedon cyclisation with simultaneous cleavage from the resin. After anappropriate peptide sequence is assembled by solid phase synthesis onthe resin or a linear sequence is appended to resin, the deprotectedamino group can react with its anchoring active linkage to produceprotected cyclic peptides. In general, a final deprotection step isrequired to yield the target cyclic peptide. The procedure forsynthesising cyclic peptides are well known in the art.

For example, lactamazation, a form of cyclisation, may be performed toform a lactam bridge using Fmoc synthesis, amino acids with differentprotecting groups at the lateral chains may be introduced, such as, butnot limited to, aspartic acid (or glutamic) protected with allyl esterat the beta ester (or gamma ester for glutamic acid) and lysineprotected with allyloxy carbamate at the N-ε. At the end of thesynthesis, with the N-terminus of the peptide protected with Fmoc, Bocor other protecting group different from Alloc, the allyl and allocprotecting groups of aspartic acid and lysine may be deprotected with,for example, palladium (0) followed by cyclization using PyAOP(7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium-hexafluorophosphate) to produce the lactam bridge.

Unless otherwise indicated, an amino acid named herein refers to theL-form. Well recognised abbreviations in the art will be used todescribe amino acids, including levoratory amino acids (L-amino acids orL or L-form) and dextrorotary amino acids (D-amino acids or D orD-form), Alanine (Ala or A), Arginine (Arg or R), Asparagine (Asn or N),Aspartic acid (Asp or D), Cysteine (Cys or C), Glutamic acid (Glu or E),Glutamine (Gln or Q), Glycine (Gly or G), Histidine (H is or H),Isoleucine (Ile or I), Leucine (Leu or L), Lysine (Lys or K), Methionine(Met or M), Phenylalanine (Phe or F), Proline (Pro or P), Serine (Ser orS), Threonine (Thr or T), Tryptophan (Trp or W), Tyrosine (Tyr or Y) andValine (Val or V). An L-amino acid residue within the native peptidesequence may be altered to any one of the 20 L-amino acids commonlyfound in proteins or any one of the corresponding D-amino acids, rareamino acids, such as, but not limited to, 4-hydroxyproline orhydroxylysine, or a non-protein amino acid, such as P-alanine orhomoserine.

Any other analogs of UAG or fragments thereof or any other modified UAGor fragments thereof that preserve the biological activity of UAG areencompassed by the present invention.

General methods and synthetic strategies used in providing functionaland structural analogs of UAG or fragments thereof are commonly used andwell known in the art and are described in publications such as “Peptidesynthesis protocols” ed, M. W. Pennigton & B. M. Dunn. Methods inMolecular Biology. Vol 35. Humana Press, NJ., 1994.

The term “homology” refers to sequence similarity between two peptideswhile retaining an equivalent biological activity. Homology can bedetermined by comparing each position in the aligned sequences. A degreeof homology between amino acid sequences is a function of the number ofidentical or matching amino acids at positions shared by the sequencesso that a “homologous sequence” refers to a sequence sharing homologyand an equivalent function or biological activity. Assessment of percenthomology is known by those of skill in the art.

Methods to determine identity and similarity of peptides are codified inpublicly available computer programs. Preferred computer program methodsto determine identity and similarity between two sequences include, butare not limited to, the GCG program package, BLASTP, BLASTN, and FASTA.The BLAST X program is publicly available from NCBI and other sources.The well known Smith Waterman algorithm may also be used to determineidentity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, J. MoI. Biol. 48: 443-453 (1970);

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992);

Gap Penalty: 12; Gap Length Penalty: 4.

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison, Wis. Theaforementioned parameters are the default parameters for amino acidsequence comparisons (along with no penalty for end gaps).

The polypeptides of the invention may be prepared in any suitable manneras known in the art. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means and methods for preparing suchpolypeptides are well known in the art.

Certain aspects of the invention use UAG polynucleotides. These includeisolated polynucleotides which encode the UAG polypeptides, fragmentsand analogs defined in the application.

As used herein, the term “polynucleotide” refers to a molecule comprisedof a plurality of deoxyribonucleotides or nucleoside subunits. Thelinkage between the nucleoside subunits can be provided by phosphates,phosphonates, phosphoramidates, phosphorothioates, or the like, or bynonphosphate groups as are known in the art, such as peptoid-typelinkages utilized in peptide nucleic acids (PNAs). The linking groupscan be chiral or achiral. The oligonucleotides or polynucleotides canrange in length from 2 nucleoside subunits to hundreds or thousands ofnucleoside subunits. While oligonucleotides are preferably 5 to 100subunits in length, and more preferably, 5 to 60 subunits in length, thelength of polynucleotides can be much greater (e.g., up to 100). Thepolynucleotide may be any of DNA and RNA. The DNA may be in any form ofgenomic DNA, a genomic DNA library, cDNA derived from a cell or tissue,and synthetic DNA. Moreover, the present invention may, in certainaspects, use vectors which include bacteriophage, plasmid, cosmid, orphagemid.

Survival Effect of UAG Fragments and Analogs Thereof

In one aspect of the invention, the proliferative and antiapoptoticeffects of UAG fragments and analogs thereof vs. UAG in INS-1E β-cellline, HIT-T15 β-cell line as well as in human pancreatic islets wereinvestigated.

UAG fragments and analogs thereof which stimulate proliferation and/orinhibit apoptosis in these cell lines will also bear other metabolicproperties of UAG including, but not limited to, lowering blood glucoselevels, improving insulin sensitivity, decreasing cortisol levels,improving lipid profile in human beings, and thus, have the potentialuse to treat metabolic disorders associated, for example, with insulinresistance, insulin deficiency, dyslipidemia or cortisol excess.

In one aspect of the invention, the survival effects of some human UAGfragments listed in Table 1 below were analyzed:

TABLE 1 SEQ ID NAME NO: SEQUENCE UAG (1-14)  2Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu- His-Gln-Arg-Val-Gln-Gln UAG (1-18)  3Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu- His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser UAG (1-5)  4 Gly-Ser-Ser-Phe-Leu UAG (17-28)  5Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys- Leu-Gln-Pro-Arg UAG (6-13)  6Ser-Pro-Glu-His-Gln-Arg-val-Gln UAG (8-13)  7 Glu-His-Gln-Arg-Val-GlnUAG (8-12)  8 Glu-His-Gln-Arg-Val UAG (8-11) 10 Glu-His-Gln-Arg UAG(9-12) 11 His-Gln-Arg-Val UAG (9-11) — His-Gln-Arg

The UAG fragments listed in Table 2 below were also analysed:

TABLE 2 SEQUENCE SEQ (amino acid residues ID 6 to 13 of NAME NO: SEQ IDNO: 1) (Asp)8 UAG (6-13)NH₂ 12 Ser-Pro-Asp-His-Gln- Arg-Val-Gln-NH₂(Lys)11 UAG (6-13)NH₂ 13 Ser-Pro-Glu-His-Gln- Lys-Val-Gln-NH₂ (Gly)6 UAG(6-13)NH₂ 14 Gly-Pro-Glu-His-Gln- Arg-Val-Gln-NH₂ (Ala)6 UAG (6-13)NH₂15 Ala-Pro-Glu-His-Gln- Arg-Val-Gln-NH₂ (Ala)7 UAG (6-13)NH₂ 16Ser-Ala-Glu-His-Gln- Arg-Val-Gln-NH₂ (Ala)8 UAG (6-13)NH₂ 17Ser-Pro-Ala-His-Gln- Arg-Val-Gln-NH₂ (Ala)9 UAG (6-13)NH₂ 18Ser-Pro-Glu-Ala-Gln- Arg-Val-Gln-NH₂ (Ala)10 UAG (6-13)NH₂ 19Ser-Pro-Glu-His-Ala- Arg-Val-Gln-NH₂ (Ala)11 UAG (6-13)NH₂ 20Ser-Pro-Glu-His-Gln- Ala-Val-Gln-NH₂ (Ala)12 UAG (6-13)NH₂ 21Ser-Pro-Glu-His-Gln- Arg-Ala-Gln-NH₂ (Ala)13 UAG (6-13)NH₂ 22Ser-Pro-Glu-His-Gln- Arg-Val-Ala-NH₂ (Acetyl-Ser)6 UAG 23Ac-Ser-Pro-Glu-His- (6-13)NH₂ Gln-Arg-Val-Gln-NH₂ (Acetyl-Ser)6, (DPro)7UAG 24 Ac-Ser-pro-Glu-His- (6-13 )NH₂ Gln-Arg-Val-Gln-NH₂ Cyclo (6-13)UAG 25 Ser-Pro-Glu-His-Gln- Arg-Val-Gln (cycl) Cyclo (8, 11), Lys 11,UAG 26 Ser-Pro-Glu-His-Gln- (6-13)amide Lys-Val-Gln-amide Cyclo (8, 11),Acetyl- 27 Ac-Ser-Pro-Glu-His- Ser6, Lys 11, UAG (6-13)- Gln-Lys-Val-Glnamide (cycl) Acetyl-Ser6, Lys 11, UAG 28 Ac-Ser-Pro-Glu-His- (6-13) NH₂Gln-Lys-Val-Gln-NH₂

UAG (1-14) and UAG (1-18) potently increased cell survival of bothINS-1E β-cells and HIT-T15 β-cells in either serum-free conditions andafter treatment with cytokines (FIGS. 1-2 for INS-1E cells, FIGS. 3A,3B, 4A and 4B for HIT-T15 β-cells). These effects were similar to thatdisplayed by the full-length molecule UAG (1-28). UAG (1-14) appearedeven stronger than native UAG as a protection against cytokine-inducedapoptosis in INS-1E cells. UAG (1-5) and UAG (17-28) exerted only atrivial effect in INS-1E cells (FIGS. 1-2) and very little effect inHIT-T15 cells (FIGS. 4A and 4B). Surprisingly, the short fragments UAG(6-13), UAG (8-13) and UAG (8-12) were all strongly effective inincreasing survival in cytokine-induced apoptosis in HIT-T15 cells (FIG.5A). Actually, peptides UAG (8-12) and UAG (8-13) were at least aspotent as UAG (1-14), whereas peptide UAG (6-13) was clearly superior.UAG (1-5) and UAG (17-28) were only minimally effective.

UAG (6-13), UAG (8-12) and UAG (8-13) were shown to exert the strongestantiapoptotic effect in HIT-T15 β-cells treated with cytokines (FIGS.6A, 6B and 6C).

The data presented herein demonstrate that UAG fragments potentlyincrease cell survival and prevent cell death in p cell lines withpotencies very comparable to that of the full-length UAG itself orbetter. UAG (1-14) exhibited a potency equivalent to, if not better thanfull-length UAG itself, whereas the (8-12) fragment, a 5 amino-acidpeptide, retained all the biological activity and UAG (6-13) was evenmore potent.

In another aspect of the invention, the data presented herein alsodemonstrate the survival effect of UAG fragments in human pancreaticislets (FIGS. 7A and 7B). UAG (1-14) and UAG (1-18) exert protectiveeffects in serum-free conditions that are similar to those displayed byUAG (1-28). On the other hand, the protective effect of UAG (1-5) and(17-28) in human islets is reduced or even absent in the experimentalconditions tested.

Effect of UAG Fragments or Analogs Thereof on Insulin Secretion

The effects of UAG (1-14) and UAG (1-18) on insulin secretion in humanislets was also investigated. UAG (1-14), similarly to UAG (1-28), andto exendin-4, significantly increased glucose-induced insulin secretionin both HIT-T15 β-cells (data not shown) and in human islets (FIGS. 8Ato 8D).

UAG Fragment and Analogs Thereof Reduce Diabetes In Vivo

In a further aspect, the data presented herein also show that UAGfragments, for example UAG (6-13), increase survival of Streptozotocin(STZ)-treated animals (FIG. 9A). UAG fragments also reduce STZ-inducedplasma glucose (FIG. 9B) and improve both plasma and pancreatic insulinlevels (FIGS. 9C and 9D) in STZ-induced diabetic rats. The datapresented herein also demonstrate that UAG fragments, for example UAG(6-13), suppress plasma glucose levels, enhance insulin sensitivity andmodulate diabetes in vivo (FIGS. 14A, 14B and 15) and reduces body fatweight (FIG. 16).

Binding of UAG Fragments and Analogs Thereof to β-Cells

In a further aspect, the data presented herein demonstrate that UAG(6-13), UAG (1-14) and UAG (1-13) recognized and bound to the UAGreceptor on HIT-T15 and INS-1E pancreatic p cells. Among these, UAG(6-13) displayed the highest binding activity and possessed a bindingaffinity very close to that of the naturally occurring UAG. Thisfinding, in conjunction with the functional in vitro studies showingthat UAG (6-13) exerts, similarly to native UAG, prosurvival effects onHIT-T15 cells, indicate that UAG (6-13) is a potent UAG agonist withpotential anti-diabetic activity.

Thus it appears that the active sequence of UAG to obtain its metaboliceffects resides in the region containing residues 8-12. This observationclearly differentiates the structure-activity relationship of UAG tothat of acylated ghrelin, for which the minimally active sequence isghrelin (1-5), the serine residue in position 3 being octanoylated. Thisfurther reinforces the hypothesis that UAG exerts its metabolic effectsthrough one or several receptors other than GHS-R1a, the receptormediating the effects of acylated ghrelin on growth hormone secretion.

Therefore, and very surprisingly, these results show that thefull-length UAG sequence is not necessary for UAG to produce itsbiological effects on β-cells and on human islet. UAG (1-14) and UAG(1-18) are at least as potent as native UAG. Even more surprisingly, UAG(8-12) and UAG (8-13) retained all the biological activity offull-length UAG, and UAG (6-13) was even more potent than UAG (1-14).

The results indicate that UAG (8-12) or any peptide comprising this 5amino acid sequence, whether amidated or not, or any peptide comprising,for example, any analogs of UAG (6-13), UAG (8-12) or UAG (8-13) willshare the same metabolic or biological effects as UAG itself. Anypeptide comprising a fragment of at least 5, or at least 6, or at least7, or at least 8 amino acid residues of the amino acid sequencecontaining residues 6 to 18 of UAG and including at least the amino acidsequence UAG (8-12) are also preferred.

In a further aspect, the present invention provides for peptidescomprising UAG (8-12) or UAG (8-13) or UAG (6-13) or any analogs thereofhaving the property to stimulate the proliferation of β-cells, toimprove survival and/or inhibit death of β-cells, to decrease plasmaglucose level, to increase insulin secretion and/or sensitivity, todecrease blood lipids, such as free fatty acids and triglycerides, toreduce cortisol secretion, to bind to β-cells, which make them useful,for example, for the treatment of disorders associated with impairedglucose metabolism, impaired insulin metabolism, type I diabetes, typeII diabetes and/or to improve the engraftment of pancreatic islets,whether by ex vivo treatment of the graft or by administration in thepatient. The peptides are also useful to treat medical conditionsassociated in insulin resistance, insulin deficiency, lower bloodglucose, useful for the treatment of diabetes, obesity and dyslipidemia.Assays for measuring the properties of the polypeptides of the inventionand the procedures for carrying out these assays are well known in theart.

In a further aspect, the present invention provides for analogs of UAGfragments which retain the biological activity of UAG. Examples of suchanalogs are, but are not limited to, (Asp) 8 UAG (6-13) where E (Glu) issubstituted by D (Asp), which is as active as UAG (6-13). The activityof this analog illustrates that a substitution of an acidic amino-acidby another acidic residue preserves the biological activity of UAG(6-13). (Lys)11 UAG (6-13) where R (Arg) is substituted by K (Lys), isalso as active as UAG (6-13), illustrating the fact that a substitutionof a basic amino-acid by another basic residue preserves the biologicalactivity of UAG (6-13). (Gly) 6 UAG (6-13) where S (Ser) is substitutedby G (Gly), is also as active as UAG (6-13), illustrating that asubstitution based on size preserves the biological activity of UAG(6-13). Overall, these analogs of UAG (6-13) demonstrate thatconservative substitutions preserve the biological activity of UAG(6-13).

Further, acetylation of Ser in position 6 (N-terminus) of UAG (6-13)preserves the biological activity of UAG (6-13) and a combination ofN-terminus acetylation and substitution of, for example, Pro7 by D-Pro(its D form) results in an analog that also exhibits biologicalactivity. Therefore, strategies aiming at stabilizing the N-terminus ofUAG (6-13) to improve its resistance to degradation by for example,exopeptidases and endopeptidases (such as, but not limited to, DPP IV)result in peptides that still exhibit biological activity of UAG (6-13),making them useful for in vivo uses.

The peptides of the present invention, including analogs thereof, can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells.Eukaryotic cells, particularly cultured cells of multicellularorganisms, are preferred. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed at least by Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, and Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY., 1987.

In general, a DNA sequence encoding the polypeptide of the presentinvention is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a polypeptide into the secretory pathway of a host cell, asecretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) may be provided in the expression vector. Thesecretory signal sequence is joined to the DNA sequence in the correctreading frame. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the propeptide of interest, although certainsignal sequences may be positioned elsewhere in the DNA sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;

Holland et al., U.S. Pat. No. 5,143,830). The methods to produce and/ormanufacture the polypeptide of the invention are

well known and well practiced in the art.

The peptides of the invention may be synthesized by solid-phasesynthesis. Solid-phase synthesis is a common method for synthesizingpeptides. Basically, in this technique, molecules are bound on a beadand synthesized step-by-step in a reactant solution; compared withnormal synthesis in a liquid state, it is easier to remove excessreactant or by-product from the product. In this method, building blocksare protected at all reactive functional groups. The two functionalgroups that are able to participate in the desired reaction betweenbuilding blocks in the solution and on the bead can be controlled by theorder of deprotection.

In the basic method of solid-phase synthesis, building blocks that havetwo function groups are used. One of the functional groups of thebuilding block is usually protected by a protective group. The startingmaterial is a bead which binds to the building block. At first, thisbead is added into the solution of the protected building block andstirred. After the reaction between the bead and the protected buildingblock is completed, the solution is removed and the bead is washed. Thenthe protecting group is removed and the above steps are repeated. Afterall steps are finished, the synthesized compound is cleaved from thebead.

If a compound containing more than two kinds of building blocks issynthesized, a step is added before the deprotection of the buildingblock bound to the bead; a functional group which is on the bead and didnot react with an added building block has to be protected by anotherprotecting group which is not removed at the deprotective condition ofthe building block. By-products which lack the building block of thisstep only are prevented by this step. In addition, this step makes iteasy to purify the synthesized compound after cleavage from the bead.

Usually, peptides are synthesized from the chain in this method,although peptides are synthesized in the opposite direction in cells. Anamino-protected amino acid is bound to a bead (a resin), forming acovalent bond between the carbonyl group and the resin. Then the aminogroup is deprotected and reacted with the carbonyl group of the nextamino-protected amino acid. The bead now bears two amino acids. Thiscycle is repeated to form the desired peptide chain. After all reactionsare complete, the synthesized peptide is cleaved from the bead.

The protecting groups for the amino groups mostly used in this peptidesynthesis are, but not limited to 9-fluorenylmethyloxycarbonyl group(Fmoc) and t-butyloxycarbonyl (Boc). The Fmoc group is removed from theamino terminus with base while the Boc group is removed with acid. Anyone of skill in the art to which this invention pertains will befamiliar with the technique of solid-phase synthesis of peptides.

Other techniques may be used to synthesize the peptides of theinvention. The techniques to produce and obtain the peptides of theinvention are well known in the art.

The peptides of the invention can be purified using fractionation and/orconventional purification methods and media. For example, ammoniumsulfate precipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable anion exchange media include derivatizeddextrans, agarose, cellulose, polyacrylamide, specialty silicas, and thelike. PEI, DEAE, QAE and Q derivatives may be used (Pharmacia,Piscataway, N.J.). Exemplary chromatographic media include those mediaderivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like.These supports may be modified with reactive groups that allowattachment of proteins by amino groups, carboxyl groups, sulfhydrylgroups, hydroxyl groups and/or carbohydrate moieties. Examples ofcoupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers.

Although UAG fragments containing amino acid residues 1-5, 1-14, 1-18,6-13, 8-12, 8-13, 8-11, 9-11, 9-12, 17-28 and analogs of UAG fragments,have been synthesized, the present invention also provides for any otherfragments of SEQ ID NO: 1 and analogs thereof retaining at least one ofthe biological activities of the full-length UAG. A skilled person inthe art, with knowledge of the instant invention, would readilydetermine if a particular UAG fragment or analog thereof has theexpected biological activities.

Therapeutic Uses and Treatments

The expression “treating a disease or a disorder” refers toadministering a therapeutic substance effective to ameliorate symptomsassociated with a disease, to lessen the severity or cure the disease,or to prevent the disease from occurring.

As used herein, the term “treatment” refers to both therapeutictreatment as well as to prophylactic and preventative measures. Those inneed of treatment include those already with the disease or disorder,condition or medical condition as well as those in which the disease,disorder, condition or medical condition is to be prevented. Those inneed of treatment are also those in which the disorder, disease,condition or medical condition has occurred and left after-effects orscars. Treatment also refers to administering a therapeutic substanceeffective to improve or ameliorate symptoms associated with a disease, adisorder, condition or medical condition to lessen the severity of orcure the disease, disorder, condition or medical condition, or toprevent the disease, disorder or condition from occurring.

The term “metabolic disorders” refers to, but is not limited to,disorders of carbohydrate metabolism, disorders of amino acidmetabolism, disorders of organic acid metabolism (organic acidurias),disorders of fatty acid oxidation and mitochondrial metabolism,disorders of porphyrin metabolism, disorders of purine or pyrimidinemetabolism, disorders of steroid metabolism, disorders of mitochondrialfunction, disorders of peroxisomal function and lysosomal storagedisorders.

The term “metabolic syndrome” refers to a combination of medicaldisorders that increase one's risk for cardiovascular disease and/ordiabetes.

It is thus an aspect of the invention that fragments of unacylatedghrelin and analogs thereof and peptides comprising them have a glucoselowering effect since unacylated ghrelin prevents the hyperglycemiceffects of acylated ghrelin, an insulin sensitizing effect, an insulinsecretion enhancement effect, a body fat weight lowering effect, a freefatty acids (FFA) and cortisol lowering effect, indicating an effect offragments of unacylated ghrelin on dyslipidemia. In addition to theseproperties, fragments of unacylated ghrelin and analogs thereof arecapable of stimulating the proliferation and the survival, as well asinhibiting death, of insulin-secreting cells such as, pancreaticβ-cells.

The invention thus provides for a therapeutic potential of fragments ofunacylated ghrelin and analogs thereof in the treatment of, for example,diabetes, other medical conditions related to impaired glucose orinsulin metabolism, insulin deficiencies or resistance, dyslipidemia,obesity, the metabolic syndrome and the treatment of insulin secretingcells such as pancreatic β-cells.

It is a further aspect, the invention provides for any pharmaceuticalcompositions incorporating at least one of the peptides of theinvention, which share the same potential therapeutic indication as UAGitself.

The peptides of the present invention can be used for and can beincorporated in pharmaceutical formulations to be used in theprevention, reduction and/or treatment of for example, but not limitedto, disorders or medical conditions associated with impaired glucosemetabolism, impaired insulin metabolism, impaired lipid metabolism, typeI diabetes, type II diabetes, obesity, dyslipidemia, atherosclerosis,cardiovascular diseases, metabolic syndrome disorders associated withimpaired proliferation of insulin-secreting cells or with insulinresistance.

For therapeutic and/or pharmaceutical uses, the peptides of theinvention may be formulated for, but not limited to, intravenous,subcutaneous, transdermal, oral, buccal, sublingual, nasal, inhalation,pulmonary, or parenteral delivery according to conventional methods.Intravenous injection may be by bolus or infusion over a conventionalperiod of time. The peptides of the invention may also be compatiblewith drug delivery system such as, but not limited to, polymer-baseddepot formulations.

Active ingredients to be administered orally as a suspension can beprepared according to techniques well known in the art of pharmaceuticalformulation and may contain, but are not limited to, microcrystallinecellulose for imparting bulk, alginic acid or sodium alginate as asuspending agent, methylcellulose as a viscosity enhancer, andsweeteners/flavoring agents. As immediate release tablets, thesecompositions may contain, but not limited to microcrystalline cellulose,dicalcium phosphate, starch, magnesium stearate and lactose and/or otherexcipients, binders, extenders, disintegrants, diluents and lubricants.

Administered by nasal aerosol or inhalation formulations may beprepared, for example, as solutions in saline, employing benzyl alcoholor other suitable preservatives, absorption promoters to enhancebioavailability, employing fluorocarbons, and/or employing othersolubilizing or dispersing agents.

The peptides of the invention may be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form. When administered byinjection, the injectable solution or suspension may be formulated usingsuitable non-toxic, parenterally-acceptable diluents or solvents,well-known in the art.

In general, pharmaceutical compositions will comprise at least one ofthe peptides of the invention together with a pharmaceuticallyacceptable carrier which will be well known to those skilled in the art.The compositions may further comprise for example, one or more suitableexcipients, diluents, fillers, solubilizers, preservatives, salts,buffering agents and other materials well known in the art dependingupon the dosage form utilised. Methods of composition are well known inthe art.

In the present context, the term “pharmaceutically acceptable carrier”is intended to denote any material, which is inert in the sense that itsubstantially does not have any therapeutic and/or prophylactic effectper se. A pharmaceutically acceptable carrier may be added to thepeptides of the invention with the purpose of making it possible toobtain a pharmaceutical composition, which has acceptable technicalproperties.

Therapeutic dose ranges of the invention will generally vary from about0.01 μg/kg to about 10 mg/kg. Therapeutic doses that are outside thisrange but that have the desired therapeutic effects are also encompassedby the present invention.

Suitable dosage regimens are preferably determined taking into accountfactors well known in the art including, but not limited to, type ofsubject being dosed; age, weight, sex and medical condition of thesubject; the route of administration; the renal and hepatic function ofthe subject; the desired effect; and the particular compound employed.

For example, a therapeutically effective amount of the peptides of theinvention (also referred to herein as “active compound”) is an amountsufficient to produce a clinically significant change in lowering bloodglucose levels, improving insulin sensitivity and/or secretion, reducingblood free fatty acids levels, lowering body fat weight, decreasingcortisol levels and/or increasing survival of insulin-secreting cells,amongst other changes. The tests for measuring such parameters are knownto those of ordinary skill in the art.

The peptides of the invention can be provided in a kit. Such a kittypically comprises an active compound in dosage form foradministration. A dosage form comprises a sufficient amount of activecompound such that a desirable effect can be obtained. Preferably, a kitcomprises instructions indicating the use of the dosage form to achievethe desired effect and the amount of dosage form to be taken over aspecified time period.

Experiments and Data Analysis UAG Fragments Promote INS-1E β-CellSurvival

Cell survival was assessed by MTT assay in INS-1E rat incubated witheither full length human UAG (1-28) or UAG (1-14), UAG (1-18), UAG (1-5)and UAG (17-28) in serum deprived medium, either alone or withIFN-γ/TNF-α/IL-1β, whose synergism has been shown to be involved inβ-cell death in both type 1 and type 2 diabetes (Ref. 16). The peptideswere tested at increasing concentrations, ranging from 0.1 nM to 100 nM.In serum-free conditions, UAG (1-14) and (1-18) showed significantsurvival effect, comparable to that of UAG (1-28). Under the sameconditions, UAG (1-5) and UAG (17-28) displayed reduced, althoughsignificant, survival action (FIG. 1). In the presence of cytokines, allthe peptides significantly increased cell survival at everyconcentration tested (FIG. 2). However, similarly to serum-freecondition, UAG (1-5) and UAG (17-28) displayed reduced effect.Interestingly, UAG (1-14) and also UAG (1-18) showed to be more potentthan full length UAG (1-28) (FIG. 2). These results indicate that UAGfragments particularly UAG (1-14) and (1-18), similarly to full lengthUAG (1-28), are able to counteract β-cell death induced by either serumstarvation or treatment with cytokines.

UAG Fragments Promote HIT-T15 β-Cell Survival

MTT experiments were also performed in hamster HIT-T15 β-cells, to testthe survival effect of UAG (1-28) or its fragments UAG (1-14), UAG(1-18), UAG (1-5) and UAG (17-28) in serum deprived medium, either aloneor with IFN-γ/TNF-α/IL-1β. As for the experiments performed on INS-1Eβ-cells, the peptides were tested at increasing concentrations, rangingfrom 0.1 nM to 100 nM. With respect to INS-1E, in HIT-T15 cells thepeptides displayed different protective effects against both serumstarvation- and cytokine-induced cell death. Indeed, whereas UAG (1-14)and UAG (1-18) significantly increased cell viability under bothexperimental conditions (FIGS. 3A and 3B), UAG (1-5) slightly increasedcell survival only in cytokine-treated cells, whereas UAG (17-28) had nosignificant effect, at any condition examined (FIGS. 4A and 4B).

The survival effect of UAG (6-13), UAG (8-13), UAG (8-12), UAG (8-11),UAG (9-12) and UAG (9-11) was assessed in cytokine-treated HIT-T15β-cells. As expected, the cytokines (IFN-γ/TNF-α/IL-1β) strongly reducedcell survival with respect to normal culture conditions (serumcontaining medium). UAG (6-13), at all the concentrations tested (1 nMto 100 nM) and particularly at 100 nM, potently inhibitedcytokine-induced cell death by increasing cell survival up to valuessimilar to or even greater than those observed in the presence of serum.Interestingly, the survival effect of UAG (6-13) was comparable to thatof full length UAG (1-28) (FIG. 5A).

Under the same experimental condition, UAG (8-13), although less thanUAG (6-13), showed significant protective effect at all theconcentrations examined, whereas UAG (8-12) displayed significant,although reduced protection, only at 10 nM and 100 nM. The protectiveeffects of peptides UAG (8-13) and UAG (8-12) were found similar tothose of UAG (1-14) and UAG (1-18) A peptide made of the inversesequence of UAG (1-14) and named UAG (14-1), was used as negativecontrol for these experiments (FIG. 5A). With regard to UAG (8-11), UAG(9-12) and UAG (9-11) (FIG. 5B), MTT results indicated that UAG (8-11)exerted significant survival effect only at 100 nM and UAG (9-12)significantly increased cell survival at both the concentrations tested(1 and 100 nM). These effects were however lower than those of UAG(6-13) (FIG. 5B). UAG (9-11) had no significant effect at bothconcentrations tested (FIG. 5B).

UAG Fragments Exert Antiapoptotic Effects in HIT-T15 β-Cells

HIT-T15 β-cells were cultured for 24 h in serum-free medium, eitheralone or with IFN-γ/TNF-α/IL-1β. In both cell lines, apoptosis increasedunder cytokine treatment, with respect to serum starvation alone. UAG(6-13) increased the number of cells, induced cell enlargement and smallislets formation, with respect to cytokine condition (data not shown).Moreover, it significantly reduced cytokine-induced apoptosis at theconcentration of 1 nM, 10 nM and, particularly, at 100 nM where theantiapoptotic effect was even stronger than that displayed by UAG (1-28)(FIG. 6A). UAG (8-13), although less than UAG (6-13), significantlyinhibited apoptosis at 10 and 100 nM, whereas UAG (8-12) showed someprotective effect only at 100 nM (FIGS. 6B and 6C respectively). UAG(14-1), the inverse sequence of UAG (1-14), was used as negativecontrol, whereas UAG (1-28), was used as positive control in eachexperiment. These results indicate that, similarly to the resultsobtained for cell survival, with respect to UAG (8-13) and UAG (8-12),UAG (6-13) exerts the strongest antiapoptotic effect in HIT-T15 β-cellstreated with cytokines.

Survival Effect of UAG Fragments in Human Pancreatic Islets

The survival effect of UAG (1-14), UAG (1-18), UAG (1-5) and UAG(17-28), with respect to that of full length UAG (1-28), was assessed inhuman pancreatic islets by MTT. The peptides were tested in islet cellscultured in serum deprived medium, either alone or withIFN-γ/TNF-α/IL-1β (5 ng/ml each). UAG (1-14) significantly increasedcell survival in serum deprived medium at 10 nM and 100 nM, whereas inthe presence of cytokines it prevented cell death at 100 nM (FIG. 7A).UAG (1-18) significantly increased cell survival at 1 nM and 10 nM (FIG.7A). UAG (1-5) displayed little, although significant survival action at10 nM in serum deprived medium but showed no cell protection afteraddition of cytokines, at any concentration tested (1 nM to 100 nM)(FIG. 7B). UAG (17-28) significantly increased survival of islet cellscultured in serum deprived conditions, at 10 nM and 100 nM, but had noeffect in the presence of cytokines (FIG. 7B). In all, these resultsindicate that in human islets, UAG (1-14) and UAG (1-18) exertprotective effects in serum-free conditions that are similar to thosedisplayed by UAG (1-28), whereas their survival capacity is at leastpartly lost in cytokine-treated cells where the effect of UAG (1-28) isstill evident.

Effect of UAG Fragments on Insulin Secretion in Human Pancreatic Islets

The effects of UAG (1-14) and UAG (1-18), both used at 100 nM, wereinvestigated on insulin secretion in human islets. FIG. 8A shows thatUAG (1-14), similarly to UAG (1-28) (FIG. 8C) and to exendin-4 (FIG.8D), significantly increased insulin secretion both in the absence andpresence of glucose (2 to 25 mM), whereas UAG (1-18) showed significanteffect with 7.5 mM glucose (FIG. 8B). UAG (1-28) and Exendin-4 were usedas positive controls (FIGS. 8C and 8D). These results indicate that inhuman pancreatic islets UAG (1-14) and UAG (1-18) stimulateglucose-induced insulin secretion.

In Vivo Effect of UAG Fragment on Streptozotocin (STZ)-Treated Animals

It is well known that Streptozotocin (STZ) treatment in neonatal ratscauses diabetes (Refs. 24, 25, 26). Herein, the long-term effects of UAG(6-13) (one week of treatment following STZ administration, assessmentat 70 days following STZ administration vs. those of UAG in neonatalrats treated with STZ at day 1 of birth) was investigated. UAG (6-13)was tested at a concentration that was equal (30 nmol/l) or higher (100nmol/l) than that of UAG. Interestingly, at day 9 after injection withSTZ, the animal survival rate, that was decreased by STZ with respect tothe Control group (≈52%), was strongly increased by UAG (≈72%), and byboth UAG (6-13) concentrations (≈71% and 89% for 30 nmol/l and 100nmol/l, respectively) (FIG. 9A). At day 70, plasma glucose wassignificantly increased by 150% (P<0.01) in STZ group with respect toControl. UAG, as expected, counteracted STZ effect by reducing glucoselevels (by ≈21%). A similar effect was obtained with both 30 nmol/l and100 nmol/l UAG (6-13) (reduction of 31% and 14%, respectively vs. STZgroup). Interestingly, UAG (6-13) at equal concentration showed aneffect that was stronger than that of UAG (FIG. 9B). STZ-treated animalsshowed significant reduction of plasma insulin levels; UAG, as well asUAG (6-13), at both concentrations, significantly reduced this effect byincreasing insulin levels in STZ-treated rats (FIG. 9C). Similar resultswere obtained with regard to pancreatic insulin secretion (FIG. 9D).These results indicate that at day 70 after treatment with STZ, UAG(6-13), similarly or even more than UAG, is able to reduce STZ-inducedplasma glucose increase and to improve both plasma and pancreaticinsulin levels.

UAG fragments modulate plasma glucose levels, insulin sensitivity aswell as gonadal fat weight in vivo in a genetic model of diabetesassociated with obesity and insulin resistance, the ob/ob mice

Baseline tail vein plasma samples were collected from free-fed and 16 hfasted ob/ob mice 7 and 6 days before pump implantation into K₂EDTAcoated capillary tubes (Microvette CB300 K2E; Sarstedt, Germany). Theanimals were then separated into three groups with approximatelyequivalent weight ranges. Ten week old mice were anesthetized, and afilled Alzet 1004 pump was inserted, delivery portal first, into theperitoneal cavity. The musculoperitoneal and skin layers were thenclosed using interrupted sutures (Vicryl 5.0 FS-2 absorbable suture).Animals received pumps containing either saline, 10 mg/ml UAG, or 3.5mg/mL UAG (6-13) (n=8 per group). Alzet 1004 pumps deliver 12 μl/day,and infused 30 μg of hUAG/animal/day (˜600 μg/kg/day) and 10 μg of UAG(6-13)/animal/day (˜200 μg/kg/day).

Blood samples (at 0900-1000) were obtained from fed and fasted animalsat weeks 2 and 4 via the tail vein into EDTA Microvette tubes. Glucoselevels in tail vein blood were measured directly using a glucometer. Onthe last day of treatment baseline (fasted) blood samples were taken.

Although no statistically significant effects (RM-ANOVA) were observedon fed plasma glucose levels during the period of treatment, UAG (6-13)showed a consistent suppressive effect relative to saline controls, andby week 4, UAG also suppressed glucose levels relative to controls (FIG.14A). In contrast, fasting glucose concentrations were significantlysuppressed by 25-30% from saline treated controls by UAG and UAG (6-13)treatment at week 2 (FIG. 14B). This effect remained at week 4 (FIG.14B). As expected, both fasting and fed glucose levels in the controlsincreased during the period of treatment, since ob/ob mice reach peakhyperglycemia at approximately 12 weeks (Ref. 27).

Fasting plasma insulin levels were significantly suppressed by UAG at 2weeks relative to saline controls (FIG. 15). By 4 weeks of treatment,though, fasting levels of insulin were significantly increased abovebaseline levels, and relative to saline controls.

During the period of treatment, in UAG (6-13) treated ob/ob animals,gonadal fat pad weight was decreased by approximately 7% relative tosaline treated controls (trend p<0.06) (FIG. 16). UAG and UAG (6-13) didnot cause an increase in gonadal fat weight over the period oftreatment, as is observed with ghrelin treatment. The trend towards adecrease in fat weight suggests that longer exposure to UAG and UAG(6-13) will exert a lipolytic effect translating into a reduction in fatmass, and thus might constitute a promising treatment for obesity, withaccompanying beneficial effects on insulin sensitivity (e.g., Refs. 28,29).

The findings from this long-term treatment protocol were that both UAGand UAG (6-13) suppressed plasma glucose levels in fasted animals after2 and 4 weeks of treatment, relative to saline control animals. UAG(6-13) also appeared to have a 30-40% suppressive effect on plasmaglucose levels in fed animals.

The effect of UAG on fasting glucose observed following 2 weeks oftreatment corresponded with significantly lowered insulin levels,indicating improved insulin sensitivity.

Binding of UAG Fragments to Pancreatic β-Cell Receptors

The ability of the fragment UAG (6-13) to compete in aconcentration-dependent manner with [¹²⁵I-Tyr⁴]-UAG for HIT-T15 (FIG.10A) and INS-1E (FIG. 10B) binding sites was assayed. As shown in FIGS.10A and 10B, unlabelled UAG (1-28) and UAG (6-13) competed with asimilar efficacy and in a concentration-dependent fashion with[¹²⁵I-Tyr⁴]-UAG for such binding sites in both cell lines. The IC₅₀values calculated from competition binding curves, all expressed as nMconcentration, were 2.6±0.5 and 2.0±0.2 for UAG (1-28) and 3.8±0.3 and2.4±0.3 for UAG (6-13) in HIT-T15 and INS-1E, respectively.

Survival Effects of UAG Fragments with Alanine Substitutions on HIT-T15β-Cells

UAG fragments with alanine (Ala) substitutions at different amino acidpositions (6 to 13) were tested with regards to their survival effectsin HIT-T15 hamster β-cells. The cells were cultured in serum deprivedmedium, either alone or with IFN-γ/TNF-α/IL-1β. The peptides were testedat the concentrations of 1 nM to 100 nM. In serum-free conditions, wherethe survival rate was reduced by ≈40% with respect to the presence ofserum, UAG (6-13) significantly increased cell survival, as expected(≈18% and ≈30% at 1 and 100 nM, respectively). Ala 6-UAG (6-13), Ala7-UAG (6-13), Ala 8-UAG (6-13), Ala 9-UAG (6-13) and particularly, Ala12-UAG (6-13) and Ala 13-UAG (6-13), showed similar effects at bothconcentrations. By contrast, very low survival effects were displayed byAla substitution at positions 10 and 11 (FIG. 11A). Under treatment withcytokines, where cell survival was reduced by ≈18% with respect to serumstarved conditions, all Ala substitutions, except those at positions 10and 11, completely reversed cell death and brought the survival rate tolevels that were even higher than those under serum-free conditions, atboth 1 nM and 100 nM concentrations. These effects, were similar tothose elicited by the original peptide UAG (6-13) (FIG. 11B). Alasubstitutions at positions 6 to 9 and 12 to 13 of UAG (6-13) do notaffect the peptide survival effect, whereas the side chains of aminoacids at position 10 (Q) and 11 (R) seem to play an essential role.

Survival Effects of UAG Fragments with Conservative Substitutions andN-Terminal Modifications on HIT-T15 β-Cells

In serum-free conditions, where the survival rate was reduced by ≈35%with respect to the presence of serum, UAG (6-13) significantlyincreased cell survival, as expected (≈18% and ≈30% at 1 and 100 nM,respectively). Asp 8-UAG (6-13), Lys 11-UAG (6-13), Gly 6-UAG (6-13), aswell as AcSer 6-UAG (6-13) and AcSer 6-(D)Pro 7-UAG (6-13) showedsimilar effects at both concentrations (FIG. 12A). Under the treatmentwith cytokines, where cell survival was reduced by ≈20%, all thepeptides significantly increased cell survival. Particularly, the besteffect was exerted by Gly 6-UAG (6-13), whereas the lowest was seenusing AcSer 6-UAG (6-13), AcSer 6-(D)Pro 7-UAG (6-13) (FIG. 12B).

Survival Effects of Cyclized UAG Fragments on HIT-T15 β-Cells

In serum-free conditions, where the survival rate was reduced by ≈58%with respect to the presence of serum, UAG (6-13) significantlyincreased cell survival, as expected (≈16% and ≈60% at 1 nM and 100 nM,respectively). Cyclo 6,13 UAG (6-13), Cyclo (8,11), Acetyl-Ser6, Lys11,UAG (6-13)amide and Acetyl-Ser6, Lys11, UAG (6-13)NH₂ showed similareffects (FIG. 13A). Similar results were found under the treatment withcytokines (FIG. 13B).

Materials and Technical Protocols

Human UAG and UAG fragments (1-14), (1-18), (1-5) and (17-28) as well asexendin-4 were from Phoenix Pharmaceuticals (Belmont, Calif.). The otherfragments (6-13), (8-13), (8-12), (8-11), (9-12), (9-11) were from TibMolBiol (Genova, Italy). Cell culture reagents were from Invitrogen(Milano, Italy). Human UAG (6-13) with alanine (Ala), Ala 6-UAG (6-13),Ala 7-UAG (6-13), Ala 8-UAG (6-13), Ala 9-UAG (6-13), Ala 10-UAG (6-13),Ala 11-UAG (6-13), Ala 12-UAG (6-13) and Ala 13-UAG (6-13) weresynthesized by Tib MolBiol (Genova, Italy).

Most of the peptides defined herein were synthesised by means of thesimultaneous multiple peptide synthesis on the following instrument:PSSM-8, SHIMADZU, Japan, using the Fmoc/But (G. Schnorrenberg et al.Tetrahedron, 45:7759, 1989) strategy by SHEPPARD (W. C. Chan et al.,Fmoc solid phase peptide synthesis—A practical approach, IRL Press,Oxford, 1989). Couplings were performed using 3-6 equiv. Fmoc-aminoacid/HOBt/TBTU and 6-12 equiv. N-Methylmorpholine on Tentagel HL RAMresin. The peptides were purified by HPLC instrument SHIMADZU LC-8A. Thepeptides were deprotected and cleaved from the resin by TFA/water andwere characterized by MALDI-TOF by means of a MALDI 2 DE instrument.Finally the peptides were lyophilized in form of the TFA salt.

Cell culture—Hamster HIT-T15 insulin-secreting β-cells were obtained andcultured as described (Refs. 14, 4). INS-1E rat β-cells were kindlyprovided by Prof. Claes B. Wollheim (University Medical Center, Geneva,Switzerland) and cultured as described (Refs. 14, 4). Cell culturereagents were from Invitrogen (Milano, Italy). Cytokines were fromBiosource (Invitrogen, Italy).

Human islet isolation—Human islets were obtained from pancreases ofmultiorgan donors as described (Ref. 4). Islet preparations withpurity >70%, not suitable for transplantation, were provided by EuropeanConsortium for Islet Transplantation (ECIT) “Islets for ResearchDistribution Program,” Transplant Unit, Scientific Institute SanRaffaele, Vita-Salute University, Milan. Islets (10,000) were culturedin CMRL (Invitrogen) with 10% FBS.

Cell survival assay—Cell survival was assessed by3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) asdescribed previously (Ref. 4). Cells were seeded on 96-well plates at adensity of 5×10³ cells/well. After treatments, cells were incubated with1 mg/ml MTT for ≈1 h. The medium was aspirated, and the formazan productsolubilized with 100 μl DMSO. Viability was assessed byspectrophotometry at 570 nm absorbance using a 96-well plate reader.

Insulin secretion—HIT-T15 cells were plated at density of 5×10⁵ cellsinto 100-mm dishes and serum starved for 24 h before incubation for 1 hat 37° C. in HEPES-buffered Krebs-Ringer bicarbonate buffer (KRBH),containing 0.5% BSA with 1.25 mM glucose. The medium was changed and thecells were incubated again for 1 h in KRBH/0.5% BSA containing 1.25, 7.5or 15 mM glucose. Following acid ethanol extraction of the hormone,secreted insulin was quantitated by a radioimmunoassay kit (LincoResearch, Labodia, Yens, Switzerland) which recognizes human insulin andcross reacts with rat insulin.

Animals—Pregnant female Sprague-Dawley rats (n=10, day 14th-15th ofpregnancy) were purchased from Harlan Sri (Italy), caged allowing freeaccess to water and fed with a standard pellet rat diet. Natural birthoccurred 6-7 days later. Five experimental groups were studied: 1)Control group, in which new-born rats received a single i.p. injectionof citrate buffer (0.05 mmol/l, pH 4.5); 2) STZ group, which received asingle i.p. injection of STZ (100 mg/Kg body weight), freshly dissolvedin citrate buffer at day 1 of birth; 3) STZ+UAG group, which received asingle i.p. injection of STZ followed by injections of UAG, (30 nmol/kgs.c., twice daily) for 7 days (from day 2 to 8) after birth; 4) STZ+UAG(6-13) group, which received a single i.p. injection of STZ followed byinjections of UAG (6-13) (30 nmol/kg s.c., twice daily) for 7 days (fromday 2 to 8) after birth; 5) STZ+UAG (6-13) group, which received asingle i.p. injection of STZ followed by injections of UAG (6-13), (100nmol/kg s.c., twice daily) for 7 days (from day 2 to 8) after birth.Dams were randomly assigned to the five groups and pups from the samelitter were assigned to the same group. The numbers of dams in each ofthe four groups 11 (Control), 11 (STZ), 16 (STZ+UAG), and 21 (STZ+UAG(6-13), 30 nmol/kg) and 15 (STZ+UAG (6-13), 100 nmol/kg). Pups were leftwith their mothers. All neonates were tested on day 2 for glycosuriausing Accu-chek compact plus (Roche). Only those animals that wereglycosuric at day 2 after birth were included in the STZ model group.Treatments with UAG and UAG (6-13) were started after glycosuria wasconfirmed. Animals were killed at day 70 after birth by decapitation.Blood samples were collected after decapitation and immediatelycentrifuged at 20,000×g for 2 min at 4° C., and stored at −20° C. untilassayed.

For the experimental data illustrated in FIGS. 14A, 14B, 15 and 16, theanimals were obtained from Charles River Laboratories (Maastricht, TheNetherlands). Animals (B6.V-Lep^(ob)/J, Charles River Laboratories,Belgian colony) were received in our animal facilities at 8 weeks ofage, and acclimatized in individual cages for 2 weeks before treatmentsbegan. They were maintained under standard 12:12 h light:darkconditions, 21° C., and were allowed free access to food and water. Theanimals were also handled daily to accustom them to the method used forblood collection. The peptides were dissolved in sterile, nonpyrogenic,0.9% saline (Baxter BV, Utrecht, The Netherlands). D-glucose wasobtained from Sigma-Aldrich Chemie BV (Zwijndrecht, The Netherlands),and was dissolved at 400 mg/ml in 0.9% saline. Alzet pumps (model 1004)were obtained from Charles River Laboratories (Maastricht, TheNetherlands). Pumps were filled with 0.9% saline, UAG or UAG (6-13)solution under sterile conditions, and pre-incubated in 0.9% saline forat least 48 hours at 37° C. to initiate flow. Blood glucose levels weremeasured directly from tail vein incisions using a Freestyle miniglucometer and test strips (ART05214 Rev.A; Abbot, Amersfoort, TheNetherlands). Plasma insulin levels were assayed by Ultra-sensitivemouse insulin ELISA (Cat. #10-1150-10; Mercodia, Sweden).

Pancreas removal and treatment—After excision, pancreases were removedand weighed. For insulin content determination, pancreases (35-50 mg)were homogenized and centrifuged in 5 ml acid-ethanol (0.15 mol/l HCl in75% [vol/vol]ethanol) at 1,000 g for 20 min; the supernatants werestored at −80° C. For immunohistochemistry, additional pancreases werefixed in 4% paraformaldehyde fixative for 24 h and embedded in paraffin.

Analytical techniques—Plasma glucose levels were determined using aglucose analyzer. Insulin was measured from pancreases or from plasma byRIA as previously described (Ref. 15).

Binding assay—Membranes from hamster HIT-T15 and rat INS-1E topancreatic β-cells were prepared and assayed for the presence of[¹²⁵I-Tyr⁴]-UAG binding. The ability of UAG fragments to compete withthe radioligand for such binding sites has been evaluated as previouslydescribed (Ref. 4). Data are presented as mean±S.E.M. of threeindependent experiments.

Statistical analysis—Results are expressed as means±SE. Statisticalanalysis were performed using Student's t test or one-way ANOVA.Significance was established when P<0.05.

It is understood that the data reported in the present specification areonly given to illustrate the invention and may not be regarded asconstituting a limitation thereof.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

All published documents mentioned in the above specification are hereinincorporated by reference.

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1. An isolated polypeptide comprising any amino acid fragment of theamino acid sequence shown in SEQ ID NO: 9 or an analog thereof, whereinsaid polypeptide has at least one activity selected from the groupconsisting of a) decreasing blood glucose levels; b) increasing insulinsecretion and/or sensitivity; c) binding to insulin-secreting cells; andd) promoting survival of insulin-secreting cells.
 2. The isolatedpolypeptide of claim 1, wherein said polypeptide comprises any aminoacid fragment of the amino acid sequence shown in SEQ ID NO: 9 or ananalog thereof, said fragment including at least the amino acid residuesshown in SEQ ID NO: 8 or an analog thereof.
 3. The isolated polypeptideof claim 1, wherein said polypeptide comprises any amino acid fragmentof the amino acid sequence shown in SEQ ID NO: 9 or an analog thereof,said fragment including at least the amino acid residues shown in SEQ IDNO: 6 or an analog thereof.
 4. The isolated polypeptide of claim 1,wherein said polypeptide comprises any amino acid fragment of at least 6amino acid residues of the amino acid sequence shown in SEQ ID NO: 9 oran analog thereof, said fragment including at least the amino acidresidues shown in SEQ ID NO: 8 or an analog thereof.
 5. The isolatedpolypeptide of claim 1, wherein said polypeptide comprises any aminoacid fragment of at least 8 amino acid residues of the amino acidsequence shown in SEQ ID NO: 9 or an analog thereof, said fragmentincluding at least the amino acid residues shown in SEQ ID NO: 8 or ananalog thereof.
 6. The isolated polypeptide of claim 1, consisting ofany consecutive 5 amino acid residues of the amino acid sequence shownin SEQ ID NO:
 9. 7. The isolated polypeptide of claim 1, wherein saidfragment has an amino acid sequence selected from the group consistingof SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:
 8. 8. An isolatedpolypeptide 5 to 27 amino acid residues in length, said polypeptidecomprising the amino acid sequence Glu-His-Gln-Arg-Val.
 9. The isolatedpolypeptide of claim 8, wherein said amino acid sequence optionallycontains up to two conservative amino acid substitutions at a positionselected from the group consisting of amino acid residues Glu, His andVal.
 10. A method for treating a disorder associated with impairedglucose metabolism in a patient, comprising administering to the patienta therapeutically effective amount of the polypeptide of claim
 1. 11.The method of claim 10, wherein the disorder is diabetes.
 12. The methodof claim 11, wherein the diabetes is type 1 diabetes.
 13. The method ofclaim 11, wherein the diabetes is type 2 diabetes.
 14. The method ofclaim 10, wherein the disorder is a medical condition associated withinsulin deficiencies.
 15. The method of claim 10, wherein the disorderis a medical condition associated with insulin resistance.
 16. Themethod of claim 10, wherein the disorder is a medical conditionassociated with dyslipidemia.
 17. The method of claim 10, wherein thedisorder is a medical condition associated with obesity.
 18. The methodof claim 10, wherein the disorder is a medical condition related tometabolic syndrome.
 19. The method of claim 10, for the treatment ofpancreatic β-cells.
 20. The method of claim 19, wherein the treatment isthrough enhancement of proliferation or of survival of the pancreaticβ-cells.
 21. The method of claim 20, wherein the enhancement ofproliferation or of survival is achieved ex vivo by subjecting thepancreatic O-cells to the polypeptide prior to administering said cellsto the patient as a graft.
 22. A method for enhancing survival and/orproliferation of insulin-secreting cells comprising culturing said cellsin the presence of a therapeutically effective amount of the polypeptideof claim
 1. 23. The method of claim 10, wherein the polypeptide isadministered through a route selected from the group consisting ofintravenous, subcutaneous, transdermal, oral, buccal, sublingual, nasaldelivery and inhalation.
 24. The method of claim 10, wherein thepolypeptide is administered in a dose varying from about 0.01 μg/kg toabout 10 mg/kg. 25.-40. (canceled)
 41. An isolated polypeptide selectedfrom the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27 and SEQ ID NO:
 28. 42. (canceled)
 43. Theisolated polypeptide of claim 41, for treating a disorder associatedwith impaired glucose metabolism in a patient.